A theoretical model of intracellular devitrification.

Devitrification of the intracellular solution can cause significant damage during warming of cells cryopreserved
by freezing or vitrification. Whereas previous theoretical investigations of devitrification have not considered
the effect of cell dehydration on intracellular ice formation, a new model which couples membrane-limited
water transport equations, classical nucleation theory, and diffusion-limited crystal growth theory is presented.
The model was used to explore the role of cell dehydration in devitrification of human keratinocytes frozen in the
presence of glycerol. Numerical simulations demonstrated that water transport during cooling affects subsequent
intracellular ice formation during warming, correctly predicting observations that critical warming rate increases
with increasing cooling rate. However, for cells with a membrane transport activation energy less than approximately
50 kJ/mol, devitrification was also affected by cell dehydration during warming, leading to a reversal of
the relationship between cooling rate and critical warming rate. Thus, for low warming rates (less than 10°C/min
for keratinocytes), the size and total volume fraction of intracellular ice crystals forming during warming decreased
with decreasing warming rate, and the critical warming rate decreased with increasing cooling rate. The
effects of water transport on the kinetics of intracellular nucleation and crystal growth were elucidated by comparison
of simulations of cell warming with simulations of devitrification in H2O–NaCl–glycerol droplets of constant
size and composition. These studies showed that the rate of intracellular nucleation was less sensitive to cell
dehydration than was the crystal growth rate. The theoretical methods presented may be of use for the design and
optimization of freeze–thaw protocols.